Adjustments of serine proteases of Daphnia pulex in response to temperature changes

Elevated temperatures considerably challenge aquatic invertebrates, and enhanced energy metabolism and protein turnover require adjustments of digestion. In Daphnia, the serine proteases chymotrypsin and trypsin represent the major proteolytic enzymes. Daphnia pulex acclimated to different temperature conditions or subjected to acute heat stress showed increased expression level of serine proteases with rising temperatures. Transcripts of trypsin isoforms were always present in higher amounts than observed for chymotrypsin. Additionally, trypsin isoform transcripts were induced by elevated temperatures to a larger extent. Correspondingly, trypsin activity dominated in cold-acclimated animals. However, the enzymatic activity of chymotrypsin increased at elevated temperatures, whereas trypsin activity slightly decreased, resulting in a shift to dominating chymotrypsin activity in warm-acclimated animals. Zymograms revealed eight bands with proteolytic activity in the range of 20 to 86 kDa. The single bands were assigned to trypsin or chymotrypsin activity applying specific inhibitors or from casein cleavage products identified by mass spectrometric analysis. The total amount of proteolytic activity was elevated with acclimation temperature increase and showed a transient decrease under acute heat stress. The contribution of the different isoforms to protein digestion indicated induction of chymotrypsin with increasing acclimation temperature. For trypsin, the share of one isoform decreased with elevated temperature, while another isoform was enhanced. Thus differential expression of serine proteases was observed in response to chronic and acute temperature changes. The observed phenotypic plasticity adjusts the set of active proteases to the altered needs of protein metabolism optimizing protein digestion for the temperature conditions experienced in the habitat. (C) 2016 Elsevier Inc. All rights reserved

Analysis of the Phosphoproteome of Chlamydomonas reinhardtii Provides New Insights into Various Cellular Pathways

The unicellular flagellated green alga Chlamydomonas reinhardtii has emerged as a model organism for the study of a variety of cellular processes. Posttranslational control via protein phosphorylation plays a key role in signal transduction, regulation of gene expression, and control of metabolism. Thus, analysis of the phosphoproteome of C. reinhardtii can significantly enhance our understanding of various regulatory pathways. In this study, we have grown C. reinhardtii cultures in the presence of an inhibitor of Ser/Thr phosphatases to increase the phosphoprotein pool. Phosphopeptides from these cells were enriched by immobilized metal-ion affinity chromatography and analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS-MS as well as neutral-loss-triggered MS-MS-MS spectra. In this way, we were able to identify 360 phosphopeptides from 328 different phosphoproteins of C. reinhardtii, thus providing new insights into a variety of cellular processes, including metabolic and signaling pathways. Comparative analysis of the phosphoproteome also yielded new functional information on proteins controlled by redox regulation (thioredoxin target proteins) and proteins of the chloroplast 70S ribosome, the centriole, and especially the flagella, for which 32 phosphoproteins were identified. The high yield of phosphoproteins of the latter correlates well with the presence of several flagellar kinases and indicates that phosphorylation/dephosphorylation represents one of the key regulatory mechanisms of eukaryotic cilia. Our data also provide new insights into certain cilium-related mammalian diseases

J-Domain Protein CDJ2 and HSP70B Are a Plastidic Chaperone Pair That Interacts with Vesicle-Inducing Protein in Plastids 1

J-domain cochaperones confer functional specificity to their heat shock protein (HSP)70 partner by recruiting it to specific substrate proteins. To gain insight into the functions of plastidic HSP70s, we searched in Chlamydomonas databases for expressed sequence tags that potentially encode chloroplast-targeted J-domain cochaperones. Two such cDNAs were found: the encoded J-domain proteins were named chloroplast DnaJ homolog 1 and 2 (CDJ1 and CDJ2). CDJ2 was shown to interact with a ∼28-kDa protein that by mass spectrometry was identified as the vesicle-inducing protein in plastids 1 (VIPP1). In fractionation experiments, CDJ2 was detected almost exclusively in the stroma, whereas VIPP1 was found in low-density membranes, thylakoids, and in the stroma. Coimmunoprecipitation and mass spectrometry analyses identified stromal HSP70B as the major protein interacting with soluble VIPP1, and, as confirmed by cross-linking data, as chaperone partner of CDJ2. In blue native-PAGE of soluble cell extracts, CDJ2 and VIPP1 comigrated in complexes of >>669, ∼150, and perhaps ∼300 kDa. Our data suggest that CDJ2, presumably via coiled-coil interactions, binds to VIPP1 and presents it to HSP70B in the ATP state. Our findings and the previously reported requirement of VIPP1 for the biogenesis of thylakoid membranes point to a role for the HSP70B/CDJ2 chaperone pair in this process

Comparative transcriptome and proteome analysis reveals a global impact of the nitrogen regulators AreA and AreB on secondary metabolism in Fusarium fujikuroi.

The biosynthesis of multiple secondary metabolites in the phytopathogenic ascomycete Fusarium fujikuroi is strongly affected by nitrogen availability. Here, we present the first genome-wide transcriptome and proteome analysis that compared the wild type and deletion mutants of the two major nitrogen regulators AreA and AreB. We show that AreB acts not simply as an antagonist of AreA counteracting the expression of AreA target genes as suggested based on the yeast model. Both GATA transcription factors affect a large and diverse set of common as well as specific target genes and proteins, acting as activators and repressors. We demonstrate that AreA and AreB are not only involved in fungal nitrogen metabolism, but also in the control of several complex cellular processes like carbon metabolism, transport and secondary metabolism. We show that both GATA transcription factors can be considered as master regulators of secondary metabolism as they affect the expression of more than half of the 47 putative secondary metabolite clusters identified in the genome of F. fujikuroi. While AreA acts as a positive regulator of many clusters under nitrogen-limiting conditions, AreB is able to activate and repress gene clusters (e.g. bikaverin) under nitrogen limitation and sufficiency. In addition, ChIP analyses revealed that loss of AreA or AreB causes histone modifications at some of the regulated gene clusters

Additional file 8: of The transcriptomic and proteomic responses of Daphnia pulex to changes in temperature and food supply comprise environment-specific and clone-specific elements

Table S2. Labeling design of the microarrays. (PDF 83 kb

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Additional file 5: of The transcriptomic and proteomic responses of Daphnia pulex to changes in temperature and food supply comprise environment-specific and clone-specific elements

Figure S4. Two-dimensional protein gel from the D. pulex clone G under heat-and-starvation stress. The 2D gel, which is a fusion (averaged) image from a varying number (n) of gels (biological replicates, 25-30 animals each), shows changes in protein expression in the D. pulex clone G after the acute exposure of control animals (20 °C, ad libitum feeding) (blue spots; n = 5) to (a) 24 h (orange spots; n = 5) of heat-and-starvation stress (T = 30 °C, starvation). (Clone G did not survive 48 h of heat-and-starvation stress.) Red or green spot IDs mark significantly up- or downregulated proteins (t-tests, P < 0.05; see Table 5). The scatter plot shows changes in expression level (Vrelative, relative spot volume) between control animals and animals exposed to heat-and-starvation stress (b, 24 h) of significantly (large circles and letters) or non-significantly (small circles) up- or down-regulated proteins (data from a). Proteins, which were upregulated under heat-and-starvation stress, are found above the diagonal line. (PDF 152 kb

Additional file 2: of The transcriptomic and proteomic responses of Daphnia pulex to changes in temperature and food supply comprise environment-specific and clone-specific elements

Figure S1. Two-dimensional protein gels from the 20 °C-acclimated D. pulex clones G and M. The RuBPs-stained 2D gels from control animals (20 °C, ad libitum feeding) of (a) the D. pulex clone G [fusion image from n = 5 gels (biological replicates)] and (b) the D. pulex clone M [fusion image from n = 4 gels (biological replicates, 25-30 animals each)] served as reference for the excision of protein spots for mass spectrometry (pI: 4.5-6, molecular mass: 20-150 or 20-100 kDa). Red characters are spot identifiers (IDs). (PDF 118 kb

Additional file 3: of The transcriptomic and proteomic responses of Daphnia pulex to changes in temperature and food supply comprise environment-specific and clone-specific elements

Figure S2. Two-dimensional protein gels from the D. pulex clone G under starvation stress. The 2D gels, which are fusion (averaged) images from a varying number (n) of gels (biological replicates, 25-30 animals each), show changes in protein expression in the D. pulex clone G after the acute exposure of control animals (20 °C, ad libitum feeding) (blue spots; n = 5) to (a) 24 h (orange spots; n = 4) or (c) 48 h (orange spots; n = 4) of starvation (T = 20 °C). Red or green spot IDs mark significantly up- or downregulated proteins (t-tests, P < 0.05; see Table 4). The scatter plots show changes in expression level (Vrelative, relative spot volume) between control and starving animals (b, 24 h; d, 48 h) of significantly (large circles and letters) or non-significantly (small circles) up- or down-regulated proteins (data from a or c). Proteins, which were upregulated under starvation stress, are found above the diagonal line. (PDF 126 kb

Additional file 7: of The transcriptomic and proteomic responses of Daphnia pulex to changes in temperature and food supply comprise environment-specific and clone-specific elements

Figure S6. Experimental design. (PDF 144 kb